It Has Become Increasingly Evident That Atheroma Development Is an Inflammatory Disease

1 CVR-2008-348R11Atherosclerosis development in apolipoprotein E-null mice deficient for CD6923Manuel Gómez1,5, Silvia M. Sanz-González2,4,5, Yafa Naim Abu Nabah2, Amalia Lamana1, 4Francisco Sánchez-Madrid1,3 and Vicente Andrés2, 6561 Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid, 7 Spain.82 Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy,9 Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, 10 Valencia, Spain.113 Departamento de Biología Vascular e Inflamación. Centro Nacional de Investigaciones 12 Cardiovasculares, Madrid, Spain.13 4 Present address: Centro de Investigación Príncipe Felipe, Valencia, Spain.145 Authors with equal contribution156 To whom correspondence should be addressed:16 Vicente Andrés, PhD17 Instituto de Biomedicina de Valencia.18 C/ Jaime Roig 11, 46010. Valencia (Spain)19 Phone: +34 963391752 Fax: +34 963391751 E-mail: [email protected]2021Short Title: CD69 and atherosclerosis22Word Count Text: 523323Word Count Abstract: 24624Number of Figures: 625Supplementary material: Yes2 1 1 CVR-2008-348R11ABSTRACT2AIMS: Atherosclerosis is a chronic inflammatory disease regulated by immune mechanisms. 3CD69 is a cell surface receptor rapidly induced after leukocyte activation at sites of chronic 4inflammation. Genetic disruption of CD69 in the mouse aggravates collagen-induced arthritis 5(CIA), and partial depletion of CD69 expressing cells with anti-CD69 monoclonal antibody 6(mAb) prevents CIA development in wild-type mice, suggesting that this receptor negatively 7modulates immune and inflammatory responses. It has been recently reported that CD69 is 8upregulated in a large subset of T cells in atherosclerosis-prone apolipoprotein E-null mice 9(apoE-/-). In this study, we investigated whether altering CD69 function affects atherosclerosis 10development. METHODS: We studied native and diet-induced atherosclerosis in apoE-/- and 11doubly deficient apoE-/-CD69-/- mice and performed expression studies in tissues and primary12cells derived from these animals. RESULTS: Plasma cholesterol level was unaffected by 13CD69 genetic inactivation. Whilst this genetic manipulation led to an elevated production of 14interferon  and interleukin 10 by activated T cells, apoE-/- and apoE-/-CD69-/- mice fed 15control and high-fat diet exhibited atheromas of similar size and composition when analyzed at16different stages of the disease. Likewise, anti-CD69 mAb treatment had no effect on plasma 17cholesterol and atherosclerosis burden in fat-fed apoE-/- mice. CONCLUSIONS: In contrast 18to previous studies highlighting the protective function of CD69 against CIA, an autoimmune 19inflammatory disease, our results rule out a significant role for CD69 against atherosclerosis in 20apoE-/- mice, an experimental disease model featuring a local inflammatory response triggered21and sustained by alterations in lipid homeostasis.2 2 1 CVR-2008-348R11INTRODUCTION23 Atherosclerosis and associated cardiovascular disease (e.g., myocardial infarction, 4stroke, and peripheral vascular disease) are the principal cause of morbidity and mortality in 5developed countries 1. Neointimal lesion formation due to the progressive accumulation of 6cellular and non-cellular elements within the subendothelial space of the artery wall is a 7hallmark of atherosclerosis. Factors contributing to neointimal cell accumulation include the 8recruitment of circulating leukocytes (mainly monocytes, which differentiate into 9macrophages, and T cells) and excessive macrophage and vascular smooth muscle cell 10(VSMC) proliferation 1-4. The activation of neointimal leukocytes triggers an inflammatory 11response characterized by abundant production of cytokines and chemokines, which lead to 12exacerbated leukocyte recruitment and proliferation and to the induction of VSMC hyperplastic13growth and migration from the tunica media towards the growing neointimal lesion 5, 6, thus 14contributing to the progression of the disease. 15 A large body of evidence supports that the inflammatory response associated to 16atherosclerosis is modulated by both adaptive and innate immune mechanisms 7-9. Monocyte-17derived macrophages, cells of the innate immune system which accumulate in plaques as 18inflammatory foam cells, play an important role in the initiation and progression of 19atherosclerosis 10, 11. Adaptive immune system cells (e.g., T and B lymphocytes and natural 20killer T cells) are also present in atheromas, with T cells being the most abundant infiltrating 21subset of lymphocytes 9. Although it is well known that murine atherosclerosis can be 22aggravated or attenuated by different subsets of T cells, our knowledge of the molecular 23mechanisms regulating the balance between pro- and anti-atherogenic immune responses is 24limited 9, 12. 2 3 1 CVR-2008-348R11 CD69 is a homodimeric leukocyte transmembrane protein that is transiently expressed 2in vitro upon cell activation 13-16. Expression of CD69 is detected in small subsets of T and B 3cells in peripheral lymphoid tissues 17. In addition, CD69 is persistently expressed in the 4infiltrates of leukocytes produced during the course of chronic inflammatory diseases (e.g., 5rheumatoid arthritis and chronic viral hepatitis) 18, 19 and autoimmune disorders (e.g., systemic 6lupus erythematosus insulin-dependent diabetes mellitus and autoimmune thyroiditis) 20-22. 7Whilst some early in vitro findings have suggested a proinflammatory function of CD69 15, 23, 8subsequent in vivo studies showed that the development of hematopoietic cells, the maturation 9of T cells and positive and negative selection of thymocytes are normal in CD69-deficient mice10(CD69-/-) 24. Remarkably, these mice show an exacerbated form of collagen induced arthritis 11(CIA) characterized by increased inflammation and augmented production of proinflammatory 12cytokines (e.g., IL-1 and RANTES) that correlate with diminished local synthesis of 13transforming growth factor 1 (TGF-1) 25. Moreover, CD69-/- mice challenged with MHC 14Class Ilo tumors exhibit reduced tumor growth and prolonged survival due to enhanced activity 15of natural killer cells and reduced production of TGF- 26. Thus, CD69 appears to be a key 16modulator of in vivo inflammatory responses in different pathological settings 27. Further 17support to this notion was provided by the observation that systemic treatment with different 18anti-CD69 monoclonal antibodies (mAb) can either exacerbate or inhibit the course of CIA in 19the mouse 28. Moreover, the ability of the anti-CD69 mAb 2.3 to reduce inflammation in CIA 20opens the possibility for its therapeutic use as an anti-inflammatory agent. 21 Altogether, the aforementioned results underscore CD69 as a negative regulator of 22autoimmune reactivity and a putative therapeutic target in inflammation. Whilst recent studies 23using atherosclerosis-prone apolipoprotein E-null mice (apoE-/-) have suggested that the 24upregulation of CD69 induces the activation of a large pool of T cells during atheroma 25development 29, it remains to be established whether CD69 expression and atherogenesis are 2 4 1 CVR-2008-348R11indeed causally linked. To address this question, we investigated in this study the development 2of atherosclerosis in apoE-/- and doubly deficient apoE-/-CD69-/- mice. Moreover, we 3examined the consequences of targeting CD69 with mAb 2.3 on atherosclerosis burden in 4apoE-/- mice. Our results show that neither spontaneously formed nor diet-induced 5atherosclerosis is altered by in vivo targeting CD69.2 5 1 CVR-2008-348R11Methods23Expanded methods are provided in Supplementary Material.42 6 1 CVR-2008-348R11Mice. The investigation conforms with the Guide for the Care and Use of Laboratory Animals 2published by the US National Institutes of Health and was approved by the ethics review board3of Consejo Superior de Investigaciones Científicas. CD69-/- mice backcrossed 12 times on a 4C57BL6 genetic background 24 and apoE-/- mice (C57BL6/J, Charles River) were mated. The 5resulting F1 was intercrossed, and F2 apoE-/- and apoE-/-CD69-/- mice were crossed to 6generate the two experimental groups (apoE-/- and apoE-/-CD69-/-). Genotyping was done by 7PCR (see sequence of primers in online supplement). 8 All studies were carried out with males. Spontaneous atherosclerosis was studied in 10-9month-old mice always fed a low-fat standard rodent diet (reference 2014, Teklad global 10rat/mouse chow, Harlan Interfauna). When stated, 3-month-old mice were switched for the 11indicated time to high-fat diet containing 0.75% cholesterol (reference S8492-E010, Ssniff). 12Four apoE-/-CD69-/- and two apoE-/- mice died during fat-feeding. Blood was withdrawn 13from the retroorbital plexus to measure plasma cholesterol level using a liquid stable reagent 14(WAKO). Cholesterol bound to high-density lipoprotein (HDL-c) and to non-HDL (non-HDL-15c) were quantified with the same reagent after precipitation with Dextran-sulphate MgCl2 16(SIGMA) as described 30.1718Anti-CD69 mAb therapy. Three groups of 2-month-old male apoE-/- mice were used. The 19control group consisted of mice fed with the standard diet throughout the study. Mice of the 20therapy groups were injected 300 g of antibody (intraperitoneally, once per week) and 21switched to atherogenic diet for three weeks. Antibody injections were repeated once per week 22during the 3-week period of fat feeding. First therapy group was injected with the anti-mouse 23CD69 mAb 2.3 (mouse IgG2a) 28 and the second therapy group received mouse IgG2a anti-24human CD4 mAb HP 2/6 as isotype-matched control (IMC). 252 7 1 CVR-2008-348R11Atherosclerosis studies. Mice were euthanized and their aortas were washed in situ with PBS 2and fixed with freshly prepared 4% paraformaldehyde/PBS. The heart and aorta were 3extracted, and fixation continued for 22-28 hours at 4ºC. Atherosclerosis was quantified by 4computer-assisted planimetry of whole-mounted aortae stained with Oil Red O (0.2% in 80% 5MeOH, SIGMA) or cross-sections stained with hematoxylin-eosin essentially as described 31, 32.6Briefly, 3-mm cross-sections were obtained from 2-3 different zones within the aortic root 7region and the ascending aorta. In both regions, the extent of atherosclerosis was quantified as 8the surface of lesion. In cross-sections of the ascending aorta, atherosclerosis was also 9quantified as the intima-to-media ratio (surface area of the intimal lesion divided by the surface10area of the media). For each mouse, the values obtained in the different sections analyzed were 11averaged. Images were captured with an Olympus CAMEDIA-C5060 wide zoom digital 12camera mounted on a Zeiss-Axiolab stereomicroscope. The Sigma Scan Pro v5.0 software 13(Jandel Scientific) was used to quantify atherosclerosis burden by an investigator who was 14blinded to genotype. Methods for the immunohistopathological characterization of atheromas 15are in Supplementary Material. 1617Statistical Analysis. Results are reported as mean ± SEM. For comparisons between two 18groups, differences were evaluated using a two-tail, unpaired t-test. For more than two groups, 19differences were evaluated using ANOVA and post-hoc Fisher's PLSD test (Statview, SAS 20Institute). Differences were considered statistically significant at p≤0.05.2 8 1 CVR-2008-348R11RESULTS23Genetic ablation of CD69 has no effect on diet-induced and spontaneous atherosclerosis 4in apoE-/- mice5The apoE-/- mouse spontaneously develops hypercholesterolemia and complex atherosclerotic 6lesions, two processes that can be accelerated by a high-fat cholesterol-rich diet 33. We first 7explored the role of CD69 on diet-induced atherosclerosis by examining 3-month-old apoE-/- 8and apoE-/-CD69-/- mice which had been challenged with an atherogenic diet for the last 7 9weeks prior to sacrifice. Pre-diet plasmatic levels of total cholesterol were similar in both 10groups of mice (apoE-/-: 26720 mg/dL, apoE-/-CD69-/-: 38116 mg/dL, p>0.05) (Fig.1A). 11As expected, fat feeding significantly elevated plasmatic cholesterol levels but no statistically-12significant differences were detected between apoE-/- and apoE-/-CD69-/- mice (134062 and 13130460 mg/dL, respectively; both p<0.0001 versus pre-diet) (Fig.1A). In concordance with 14previous studies in the apoE-/- mouse model, the increase in plasma cholesterol induced by fat 15feeding was mainly contributed by non-HDL-cholesterol, which was similar in both groups of 16mice (apoE-/-: 131662 mg/dL, apoE-/-CD69-/-: 127259 mg/dL; both p<0.0001 versus pre-17diet) (Fig.1A). We also found no effect of CD69 disruption on atherosclerosis burden within 18the aortic arch and thoracic aorta, as assessed by Oil Red O staining of whole-mounted vessels 19(Fig.2A). Consistent with these findings, analysis of the intima-to-media ratio and total lesional20surface in cross-sections from the ascending aorta (Fig.2C) and aortic root (Fig.2E) revealed 21similar extent of atherosclerosis when comparing apoE-/- and apoE-/-CD69-/- mice challenged 22with the atherogenic diet for 7 weeks. Moreover, atherosclerosis burden was not affected by 23CD69 deletion in apoE-/- mice fed atherogenic diet for 2 weeks, which developed 24comparatively smaller aortic atheromas (Fig.S1, online supplement).2 9 1 CVR-2008-348R11 It was feasible that a possible subtle effect of CD69 disruption on atherosclerosis might 2have been masked under highly atherogenic conditions, such as occur in severely 3hypercholesterolemic fat-fed apoE-/- mice. However, 10-month-old apoE-/- and apoE-/-4CD69-/- mice fed standard chow, which exhibited plasmatic total cholesterol <400 mg/dL 5(Fig.1B), developed atheromas of similar size within the aortic arch and thoracic aorta 6(Fig.2B), ascending aorta (Fig.2D), and aortic root region (Fig.2F). Immunohistopathological 7examination of atherosclerotic lesions revealed undistinguishable neointimal accumulation of 8Mac-3-immunoreactive macrophages, SM-actin-immunoreactive VSMCs, CD4-9immunoreactive T cells and collagen when comparing apoE-/- and apoE-/-CD69-/- mice fed 10either standard chow (Fig.3A) or atherogenic diet (Fig.3B). Collectively, these studies 11demonstrate that both the size and composition of spontaneously-formed and diet-induced 12aortic atheromas are unaffected by genetic disruption of CD69 in apoE-/- mice when analyzed 13at different stages of the disease. 14 Next, we studied the production of effector cytokines by activated T lymphocytes from 15apoE-/- and apoE-/-CD69-/- mice. Secreted cytokines were analyzed by ELISA in the 16supernatant of cultured splenic cells which were activated in vitro with mAb anti-CD3. These 17studies revealed elevated levels of both the proinflammatory Th1-type cytokine IFN- and the 18immunosuppressive cytokine IL10 in apoE-/-CD69-/- compared with apoE-/- cells, with the 19Th2-type cytokine IL4 being unaffected by CD69 ablation (Fig.4A). Thus, despite the lack of 20effect on atheroma formation, CD69 genetic inactivation in apoE-/- mice affects at least some 21aspects of the immune reactivity of T lymphocytes. We also investigated by quantitative real-22time RT-PCR (qPCR) the expression of CD25, MHC class II and the chemokine receptors 23CCR7 and CXCR4 in the spleen of mice fed control diet (Fig.4B) or challenged for 6 weeks 24with the atherogenic diet (Fig.4C). The results of these studies did not reveal statistically-25significant differences between groups, thus suggesting that a compensatory upregulation of 2 10 1 CVR-2008-348R11leukocyte activation molecules in fat-fed apoE-/-CD69-/- mice is unlikely to account for the 2lack of effect of CD69 inactivation on atheroma development. 3 Since macrophages play an important role in the initiation and progression of 4atherosclerosis and upregulate CD69 upon activation 34, we assessed whether CD69 ablation 5affects the inflammatory state of macrophages. To this end, we analyzed by qRT-PCR the 6expression of the proinflammatory cytokines TNF- and IFN- in peritoneal macrophages 7harvested from mice fed control diet. As shown in Fig.4D, both cytokines were expressed at 8similar levels in apoE-/- and apoE-/-CD69-/- mice.910In vivo treatment with the depleting anti-CD69 mAb 2.3 has no effect on atherosclerosis 11burden in apoE-/- mice12We previously showed that CD69 targeting by mAbs affects the course of CIA in the mouse 28. 13In particular, treatment with anti-CD69 mAb-2.3 reduced the severity of CIA by partially 14depleting CD69-expressing effector cells. To investigate whether this mAb also protects 15against atherosclerosis, we studied three groups of apoE-/- mice: untreated group fed control 16diet, and fat-fed mice which were treated with either anti-CD69 mAb-2.3 or with mAb-isotype-17matched control (IMC) (intraperitoneal administration, once per week during the 3-week 18period of fat-feeding). We first performed flow cytometric analysis of thymocytes isolated 19from mAb-treated groups and stained ex vivo with an anti-CD69 mAb different from 2.3. As 20shown in Fig.5A, in vivo treatment with mAb-2.3 greatly reduced CD69 expression in 21thymocytes as compared with cells from mice treated with control mAb-IMC, thus confirming 22that mAb-2.3 binds in vivo to CD69 expressing cells. We also examined the production of 23effector cytokines by splenic cells activated in vitro with anti-CD3 mAb (Fig.5B). These 24experiments disclosed no statistically significant differences in the production of IFN-, IL4 25and IL10 when comparing the treatments with mAb-2.3 and mAb-IMC.2 11 1 CVR-2008-348R11 Compared with basal levels in untreated mice fed control diet, plasmatic total cholesterol 2was similarly elevated in both groups of fat-fed mAb-treated mice (Fig.6A, both p<0.0001 3versus control diet). Likewise, atherosclerosis burden was similar in mAb-2.3- and mAb-IMC-4treated mice, as revealed by Oil Red O staining of the aortic arch and thoracic aorta (Fig.6B) 5and quantification of the intima-to-media ratio and total lesion size in cross-sections through 6the ascending aorta (Fig.6C). Moreover, the area occupied by neointimal macrophages in 7atheromas from the aortic root was similarly increased in both groups of fat-fed mAb-treated 8mice compared with controls fed standard chow (Fig.6D). Taken together, these studies 9demonstrate that treatment with the anti-CD69 mAb-2.3 has no effect on the extent of 10atherosclerosis in fat-fed apoE-/- mice.111213DISCUSSION14 Atherosclerosis is a chronic inflammatory disease modulated by both adaptive and 15innate immune mechanisms 7, 8. It is well known that different subsets of T-cells can exacerbate16or attenuate murine atherosclerosis 7-9, 12, therefore understanding the molecular mechanisms 17that regulate the balance between pro- and anti-atherogenic immune responses is of major 18importance. We have previously shown that CD69-/- mice display an exacerbated form of CIA,19suggesting that CD69 is a negative regulator of inflammatory responses 25, 27. Notably, the 20percentage of lymphocytes expressing both CD69 and CD3 is 2-3 times higher in apoE-/- mice 21as compared with wild-type controls, suggesting that atherosclerosis may activate a large pool 22of T cells through CD69 upregulation 29. This could also be true for monocytes/macrophages, 23although expression of CD69 in these cells has not been characterized in the context of 24atherosclerosis. Importantly, dietary factors seem to modulate the immune response associated 25to atherogenesis. For example, the absence of T and B cells had no effect on the extent of 2 12 1 CVR-2008-348R11atherosclerosis in apoE-/- mice fed for 12 weeks a fat- and cholesterol-enriched diet, but 2decreased atherosclerosis burden in apoE-/- mice fed standard chow 35, 36. Therefore, in this 3study we have examined atherosclerotic lesion formation in apoE-/-CD69-/- doubly deficient 4mice and in apoE-/- controls receiving either control chow or a high-fat diet to investigate the 5role of CD69 on both spontaneous and diet-induced atherosclerosis, respectively. In both 6experimental settings, neither atherosclerosis burden nor lesion composition was altered by 7CD69 disruption. Of note in this regard, previous analysis of CD69-/- mice did not reveal 8differences in the phenotype of lymphocyte populations or in the expression of activation 9markers such as CD25 and CD44 24 (and unpublished results). In the present study, we found 10that the mRNA level of CD25, MHC class II, and chemokine receptors CCR7 and CXCR4 is 11not significantly altered in the spleen of apoE-/-CD69-/- mice fed either control or atherogenic 12diet (Fig.4B,C), suggesting that the lack of effect of CD69 ablation on atherosclerosis is not 13due to a compensatory upregulation of other immunomodulators. Moreover expression of the 14proinflammatory cytokines IFN- and TNF- in peritoneal macrophages harvested from 15apoE-/- mice fed control diet is not affected by CD69 deficiency, suggesting that CD69 is not 16an important player of macrophage activation in this disease model. Taking together, our 17results demonstrate that CD69 deficiency does not affect the course of spontaneous and diet-18induced atherosclerosis. 19 In the CIA model, inflammation is triggered by immunization of mice with type II 20collagen, which generates T- and B-cell responses to collagen that are crucial for disease 21development 37. Indeed, the aggravation of CIA in CD69-/- mice immunized with collagen is 22associated with exacerbated responses of T- and B-cell and enhanced inflammation 25. 23Recently, an effect of CD69 on the differentiation of T lymphocytes to effector cells has been 24proposed as one of the putative mechanisms responsible for its negative regulatory effect on 25inflammation during CIA 28. T cells also play crucial roles on atherosclerosis by controlling 2 13 1 CVR-2008-348R11inflammatory responses through the production of a plethora of regulatory cytokines 7-9, 12. Of 2particular relevance for the results reported herein, studies using genetically-modified mice 3have shown that IFN- exerts proatherogenic actions 38, whereas IL10 expression protects from4atherosclerosis 39, 40. We found similar number of infiltrating lymphocytes in atherosclerotic 5lesions of fat-fed apoE-/- and apoE-/-CD69-/- mice (Fig 3B), but secretion of both IFN-and 6IL10 was significantly elevated in T cells that were isolated from fat-fed apoE-/-CD69-/- and 7stimulated ex vivo through their T-cell receptor, as compared to apoE-/- control cells (Fig.4A). 8These results suggest that genetic ablation of CD69 in the mouse increases the immune 9reactivity of T-cells in a manner that leads to the combined upregulation of proatherogenic 10(e.g., IFN-and antiatherogenic (e,g., IL10) factors, thus providing a potential mechanism 11contributing to comparable atherosclerosis development in apoE-/-CD69-/- and apoE-/- mice. 12It is also noteworthy that T and B cells have been proposed to play a minor role in 13atherosclerosis in fat-fed apoE-/- mice, suggesting another possible explanation for the lack of 14effect of CD69 ablation on disease progression under highly atherogenic conditions. Therefore,15although our results suggest that CD69 may play a role in the regulation of T-cell 16differentiation in vivo, as previously proposed 27, additional studies in other well-dissected 17experimental systems of T-lymphocyte effector differentiation are needed to clarify this point 18(e.g., antigen-dependent differentiation of lymphocytes from T-cell receptor transgenic mice 19deficient for CD69).20 Our previous studies in the murine CIA model showed that anti-CD69 mAb-2.3 21administration partially depleted CD69 expressing activated effector T cells, including IFN-22+CD4+ lymphocytes, a response which was accompanied by a significant reduction in both 23macroscopic inflammation and the production of proinflammatory cytokines, and by an 24amelioration of disease progression 28. Thus, targeting in vivo CD69 expressing leukocytes by 25treating with mAb-2.3 might be proposed as a suitable therapeutic agent against inflammatory 2 14 1 CVR-2008-348R11processes. In the present work, we examined atherosclerosis development in apoE-/- mice 2treated with mAb-2.3 once per week during a 3-week period of fat feeding. Consistent with our3previous findings 28, we observed that mAb-2.3 bound in vivo to CD69-immunoreactive cells, 4as CD69 expression was reduced in ex vivo stained thymocytes isolated from mAb-2.3- 5compared to mAb IMC-treated mice (Fig.5A). However, mAb-2.3 did not alter in splenic cells 6the production of cytokines known to affect atherosclerosis development (e.g., IFN-, IL4 and 7IL10) (Fig.5B), and failed to exert any significant effect on atheroma formation and neointimal8macrophage accumulation in fat-fed apoE-/- mice (Fig.6). These findings suggest that mAb-2.39treatment in apoE-/- mice does not significantly deplete effector T cells, either because 10exhaustion of these cells is not taking place or the effect is too subtle to alter disease 11progression. The latter might be the consequence of a relatively low upregulation of CD69 and 12mild level of T-cell activation locally generated in response to antigens present within the 13plaques of apoE-/- mice. In contrast, the degree of T-cell activation and the level of CD69 14expression in mice immunized with collagen plus complete Freund´s adjuvant to induce CIA is15expected to be comparatively much higher, thus favouring the depleting effect of mAb-2.3 and 16the inhibitory effect on inflammation observed in this model. Although additional studies are 17required to clarify this important point, our findings seem to rule out the use of mAb-2.3 as a 18therapy against atherosclerosis. 19 In summary, contrary to previous studies that highlight a protective role of CD69 20against CIA, an autoimmune inflammatory disease, we have shown here that CD69 genetic 21ablation does not affect the size and composition of aortic atherosclerotic lesions in the apoE-/-22mouse, an experimental model in which local inflammation within the artery wall is triggered 23and sustained by alterations in lipid homeostasis. Moreover, in contrast to the beneficial effect 24of anti-CD69 mAb-2.3 against CIA, administration of this antibody does not attenuate 2 15 1 CVR-2008-348R11atherosclerosis development in apoE-/- mice. We therefore conclude that CD69 is not a good 2therapeutic target in the setting of atherosclerosis.345FUNDING6Work supported by Instituto de Salud Carlos III - Ministerio de Sanidad y Consumo of Spain 7(ISCIII-MSC) (Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares8–RECAVA, and BEFI predoctoral fellowship to SMS-G), Ministerio de Educación y Ciencia 9of Spain and European Regional Development Fund (grants SAF2004-03057 and SAF2007-1062210 to VA, and BFU2005-08435/BMC to FS-M). MG is an investigator of Programa Ramón11y Cajal 2002 from Ministerio de Ciencia y Tecnología of Spain and was supported by grant 12PI06/0937 from ISCIII-MSC.1314ACKNOWLEDGEMENTS15We thank María J. Andrés-Manzano for helping with the preparation of figures and 16morphometric analysis, and Angela Vinué for excellent technical assistance. 171819CONFLICT OF INTEREST20None.2 16 1 CVR-2008-348R11REFERENCES21. Lusis AJ. Atherosclerosis. Nature 2000;407:233-241.32. Ross R. 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Daugherty A, Pure E, Delfel-Butteiger D, Chen S, Leferovich J, Roselaar SE et al.4 The effects of total lymphocyte deficiency on the extent of atherosclerosis in 5 apolipoprotein E-/- mice. J Clin Invest 1997;100:1575-1580.637. Myers LK, Rosloniec EF, Cremer MA, Kang AH. Collagen-induced arthritis, an 7 animal model of autoimmunity. Life Sci 1997;61:1861-1878.838. Harvey EJ, Ramji DP. Interferon-gamma and atherosclerosis: pro- or anti-9 atherogenic? Cardiovasc Res 2005;67:11-20.1039. Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, Bureau MF et al. 11 Protective role of interleukin-10 in atherosclerosis. Circ Res 1999;85:e17-24.1240. Pinderski Oslund LJ, Hedrick CC, Olvera T, Hagenbaugh A, Territo M, Berliner 13 JA et al. Interleukin-10 blocks atherosclerotic events in vitro and in vivo. 14 Arterioscler Thromb Vasc Biol 1999;19:2847-2853.15162 21 1 CVR-2008-348R11FIG.1: Plasmatic cholesterol level in apoE-null mice is unaffected by CD69 inactivation. 2Quantification of plasmatic levels of total cholesterol, HDL-cholesterol (HDL-c), and non-HDL-3c in: (A) mice fed atherogenic diet for 7 weeks (n=7 mice each genotype; pre- and post-diet 4values), (B) mice fed control chow (n=4 mice each group). *, p<0.0001 versus pre-diet, same 5genotype. 67FIG.2: CD69 genetic disruption has no effect on atherosclerosis burden. Atherosclerotic lesion8size was quantified in mice fed atherogenic diet for 7 weeks (A, C, E) or control diet (B, D, F). 9(A, B) Atherosclerosis quantified in Oil Red O-stained aorta as % of surface occupied by lesion 10(red staining). Representative images are shown. The discontinuous line marks the separation 11between the aortic arch and thoracic aorta. (C, D) Atherosclerosis quantified as the intima-to-12media ratio and lesional surface in hematoxylin-eosin-stained cross-sections obtained from three 13independent zones of the ascending aorta region adjacent to the aortic valves. (E, F) 14Atherosclerosis quantified as lesional surface in hematoxylin/eosin-stained cross-sections 15obtained from three independent zones from the aortic root. For each mouse, the values obtained 16in the different cross-sections analyzed were averaged.1718FIG.3: CD69 genetic disruption has no effect on the composition of spontaneously-formed 19and diet-induced atheromas. Aortic cross-sections were obtained from mice fed control chow 20(A) or challenged with atherogenic diet for 6-7 weeks (B). Neointimal macrophages, VSMCs 21and T-cells were identified using anti-Mac-3, anti-SM-actin and anti-CD4 antibodies, 22respectively. Neointimal collagen content was quantified by Massson’s Trichrome staining. 23Tissue was embedded in paraffin (anti-Mac-3, anti- SM-actin and collagen, control chow or 24atherogenic diet for 7 weeks) or Cryoblock (anti-CD4, atherogenic diet for 6 weeks). In the 2 22 1 CVR-2008-348R11confocal microscopy studies to detect CD4-immunoreactive cells (green signal), nuclei were 2counterstained with Topro-3 (red signal). Representative images are shown.34FIG.4: Effect of CD69 ablation on cytokine production by activated splenic T cells and 5peritoneal macrophages and on the expression of activation molecules in spleen. (A) 6Splenocytes obtained from mice fed atherogenic diet for 7 weeks were resuspended in RPMI-71640 medium and activated in vitro with anti-CD3 antibody (20 g/ml). After 48 hours, 8supernatants were collected for ELISA quantification of IFN-, IL4 and IL10. (B-D) qRT-PCR 9of RNA isolated from spleen of mice fed control diet or challenged for 6 weeks with atherogenic 10diet, and from peritoneal macrophages of mice fed control diet. Results are normalized with 11GAPDH (A.U.: arbitrary units). 1213FIG.5: Anti-CD69 mAb 2.3 binds in vivo to CD69 expressing cells but has no effect on 14cytokine production by splenic T-cells. apoE-/- mice were treated with either anti-CD69 mAb-152.3 or with mAb-IMC (intraperitoneally, once per week) during a 3-week period of fat-feeding. 16(A) Percentage of CD69-expressing cells in total thymocytes determined by flow cytometry (n=417mice per group). The histograms show representative examples and the graph shows the average 18percentage of CD69+ cells. Note that mAb-2.3 binds in vivo to CD69-expressing thymocytes 19compared with mAb-IMC. (B) Splenocytes were activated in vitro with anti-CD3 antibody and 20culture supernatants were collected for ELISA quantification of IFN-, IL4 and IL10. 2122FIG.6: Effect of the anti-CD69 mAb 2.3 on atheroma development. Three different groups of 23apoE-/- mice were studied: control diet untreated (striped bars), and treated with either anti-24CD69 mAb-2.3 (black bars) or with mAb-IMC (white bars) during a 3-week period of fat-25feeding (mAb given intraperitoneally, once per week). (A) Total plasma cholesterol level. (B) 2 23 1 CVR-2008-348R11Atherosclerosis in the aortic arch and thoracic aorta quantified as the ratio of total lesion area 2versus total area in whole-mounted tissue stained with Oil Red O. Results are presented relative 3to aortic arch lesion burden in apoE-/- mice (=1). (C) Atherosclerosis quantified as the intima-to-4media ratio and lesion size measured in cross-sections of the ascending aorta. (D) Area of 5atheroma occupied by macrophages as revealed by Mac-3 immunostaining in cross-sections 6from the aortic root. In C and D, three and two independent cross-sections were measured for 7each mouse, respectively, and average values were obtained for each mouse. Differences versus 8untreated mice fed control diet: *, p<0.02; **, p<0.009; † p< 0.003; ††, p<0.0001. None of the 9parameters analyzed disclosed statistically significant differences when comparing mAb-treated 10groups.2 24

It Has Become Increasingly Evident That Atheroma Development Is an Inflammatory Disease

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